Bibliographic details of the publications referred to hereinafter in the specification are collected at the end of the description. SEQ ID No's referred to herein in relation to nucleotide and amino acid sequences are defined after the Bibliography.
The flower industry strives to develop new and different varieties of flowering plants. An effective way to create such novel varieties is through the manipulation of flower colour and classical breeding techniques have been used with some success to produce a wide range of colours for most of the commercial varieties of flowers. This approach has been limited, however, by the constraints of a particular species' gene pool and for this reason it is rare for a single species to have a full spectrum of coloured varieties. For example, the development of blue varieties of the major cut flower species such as rose, chrysanthemum, carnation, lily, tulip and gerbera would offer a significant opportunity in both the cut flower and ornamental markets.
The colours of flowers and other plant parts are predominantly due to two types of pigments: flavonoids and carotenoids. Flavonoids are the most common and the most important of the flower pigments. The most important classes of flavonoids with respect to flower colour are anthocyanins, flavonols and flavones. Anthocyanins are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and are localised in the vacuole.
One important factor for flower colour is co-pigmentation of arithocyanins with tannins and certain flavone and flavonol glycosides (Scott-Moncrieff, 1936). When compared over a range of pH values, co-pigmented anthocyanins are always found to be bluer than the normal pigment. Co-pigmentation of anthocyanins with flavonol glycosides can also be important for the development of colour in fruit (Yoshitama et al., 1992). The molar ratio of anthocyanin to co-pigment can also exert a strong influence on colour. It has recently been demonstrated that flavonol aglycones are essential for pollen germination and pollen tube growth (Mo et al., 1992). The ability to control the production of co-pigments, such as flavonols, in plants could therefore have useful applications in altering flower colour and manipulating plant fertility.
The biosynthetic pathway for the anthocyanin pigments is well established (Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979; Wiering and de Vlaming, 1984; Schram et al., 1984; Stafford, 1990). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA with one molecule of p-coumaroyl-CoA. This reaction is catalysed by the enzyme chalcone synthase. The product of this reaction, 2', 4, 4', 6'-tetrahydroxychalcone, is normally rapidly isomerised to produce naringenin by the enzyme chalcone-flavanone isomerase. Naringenin is subsequently hydroxylated at the 3 position of the central ring by flavanone 3-hydroxylase to produce dihydrokaempferol (DHK). The B-ring of dihydrokaempferol can be hydroxylated at either the 3', or both the 3' and 5' positions, to produce dihydroquercetin (DHQ) and dihydromyricetin (DHM), respectively. DHK, DHQ and DHM may be converted to coloured anthocyanins (pelargonidin 3-glucoside, cyanidin 3-glucoside and delphinidin 3-glucoside) by the action of at least two enzymes (dihydroflavonol-4-reductase and flavonoid-3-glucosyltransferase).
Flavonols such as kaempferol (K), quercetin (Q) and myricetin (M) are formed from dihydroflavonols by the introduction of a double bond between C-2 and C-3 (Forkmann, 1991), as illustrated in FIG. 1. Flavonols often accumulate in glycosylated forms and may also be methylated. Methylation can occur either before or after glycosylation. In vitro conversion of dihydroflavonols to flavonols was first observed in enzyme preparations from parsley cell cultures (Britsch er al., 1981). Flavonol synthase activity has also been detected in flower extracts from Matthiola (Spribille and Forkmann, 1984), Petunia (Forkmann et al., 1986) and Dianthus (Forkmann, 1991). Flavonol synthase enzyme activity requires 2-oxoglutarate, ascorbate and ferrous ions as cofactors. In flowers of Petunia hybrida, the genetic locus Fl controls the formation of flavonols: flavonol synthesis is greatly reduced in mutants homozygous recessive for this gene (Wiering et al., 1979; Forkmann et al., 1986). In vitro enzyme assays with the flavonol synthase from petunia showed that DHK and DHQ were readily converted to the respective flavonols, whereas DHM was a poor substrate. The ability to control flavonol synthase activity in flowering plants would provide a means to manipulate petal colour by altering flavonol production, thereby enabling a single species to express a broader spectrum of flower colours. As stated above, the ability to control flavonol production also has implications in respect of male fertility. Such control may be by modulating the level of production of an indigenous enzyme or by introducing a non-indigenous enzyme.